Analysis Of Binding Characters And Localization In Tissues Of Odorant Binding Proteins In Aphis Glycines (Matsumura)

Acute olfaction allows insect to orientate food and mates, stimulus insect hehavioral responses, and hide from predators quite accurately by discriminating semiochemical cues in natural environment. Olfactory system not only plays a crucial role in insect survival and reproductive success, but has been developed as an important way for pest control strategies. Odorant binding proteins (OBPs) are the first functional proteins which involved in the olfactory perception. Soybean aphid (Aphis glycines Matsumura) is one of the most important pests on soybean which mainly control by chemical in the past, but with developing of resistance rapidly and need of food safe on agriculture, it is very important for people to find a new way to control.According to the known pea aphid (Acyrthosiphon pisum Harris) sequences, ten OBPs genes with complete coding sequence were obtained from Aphis glycines by RT-PCR and RACE technique, and they were deposited in GenBank (AglyOBP2, KF923843; AglyOBP3, KF923844; AglyOBP4, KF923845; AglyOBP5, KF923846; AglyOBP6, KF923847; AglyOBP7, KF923848; AglyOBP8, KF923849; AglyOBP9, KF923850; AglyOBP10, KF923851; AglyOBP11, KF923852). All the AglyOBPs were predicted to contain a highly conserved domain separately, and belong to three different odorant binding protein families (Plus-C OBPs, Typical OBPs and Minus-C OBPs). Consistent evolutionary trend of orthologous genes were observed from the phylogenetic tree which constructed by aphid OBPs available for A. glycines compared with A. pisum and Aphis gossypii Glover, which indicated that some particular relationships might exist between OBP genes and selective pressure from host surroundings.Six expression plasmids which contained AglyOBP2, AglyOBP3, AglyOBP4, AglyOBP7, AglyOBP8 and AglyOBP9 separately were reconstructed and were expressed in Escherichia coli (Transetta (DE3)) host cells by inducing. Fusion proteins with six histidine tags were easily purified by the immobilized metal nickel chelate affinity chromatography, and western blotting confirmed that fusion proteins were expressed totally. The histidine tags were digested by the recombinant enterokinase (rEK), and the rest part of fusion proteins were quite close to the natural state of AglyOBPs. Fluorescence competition binding assays were applied to explore the binding characters of AglyOBPs for host soybean volatiles and aphid alarm pheromone ((E)-β-farnesene (EBF)). The six AglyOBPs showed a broad binding character for the small odorants with C6～C8 when they competed 1-NPN. AglyOBP2 was able to bind several components of host soybean volatiles which were released from the’Dongnong 46’soybean crops at micromolar level, and higher affinities exhibited when binding alcohols, aldehydes and esters with small molecular weight, just like 2-methyl-4-pentenal and some odorants belonging to green leaf volatiles (c/s-3-hexen-l-ol. trans-2-hexen-l-ol and cis-2-hexen-1-ol). which suggested that AglyOBP2 might play an important role in sensing host plant volatiles. Meanwhile. AglyOBP7 could bind trans-β-farnesene very strongly (Ki= 1.6 μM), which indicated that AglyOBP7 was an important functional protein in recognizing aphid alarm pheromone.The expression and localization in different developmental stages and tissues of six genes (AglyOBP2. AglyOBPS. AglyOBP4, AghOBP7. AglyOBP8 and AglyOBP9) were investigated by real-time fluorescent quantitative PCR. The result showed that six AglyOBPs were expressed in all developmental stages with rather small differences, and the expression increased and peaked in the 4th nymphal period, and then it dropped back to the former amount in the adult period. Expect AglyOBP7. the other five AglyOBPs were expressed higher in the alate adults than the apterous ones, which revealed that important roles might exist in the host-seeking of the alate adults. Additionally, six AglyOBPs were expressed in antennae with different expression levels. The expressions of AglyOBP2, AglyOBP7 and AglyOBP9 in antennae were significantly higher than the other tissues, and these genes were thought to be involved in olfactory recognition. Otherwise, the overexpressions of AglyOBP3, AglyOBP4 and AglyOBP8 were observed expect antennae (head tissue not including antenna, foot tissue and wing), which implied special olfactory functions they might have. It is very important of AglyOBPs expression in the head, foot and wing which can help insect recognize the odor molecules by increasing effective recognition.